title: Delineation of key XRCC4/Ligase IV interfaces for targeted disruption of non-homologous end joining DNA repair.
abstract: Efficient DNA repair mechanisms frequently limit the effectiveness of chemotherapeutic agents that act through DNA damaging mechanisms. Consequently, proteins involved in DNA repair have increasingly become attractive targets of high-throughput screening initiatives to identify modulators of these pathways. Disruption of the XRCC4-Ligase IV interaction provides a novel means to efficiently halt repair of mammalian DNA double strand break repair; however; the extreme affinity of these proteins presents a major obstacle for drug discovery. A better understanding of the interaction surfaces is needed to provide a more specific target for inhibitor studies. To clearly define key interface(s) of Ligase IV necessary for interaction with XRCC4, we developed a competitive displacement assay using ESI-MS/MS and determined the minimal inhibitory fragment of the XRCC4-interacting region (XIR) capable of disrupting a complex of XRCC4/XIR. Disruption of a single helix (helix 2) within the helix-loop-helix clamp of Ligase IV was sufficient to displace XIR from a preformed complex. Dose-dependent response curves for the disruption of the complex by either helix 2 or helix-loop-helix fragments revealed that potency of inhibition was greater for the larger helix-loop-helix peptide. Our results suggest a susceptibility to inhibition at the interface of helix 2 and future studies would benefit from targeting this surface of Ligase IV to identify modulators that disrupt its interaction with XRCC4. Furthermore, helix 1 and loop regions of the helix-loop-helix clamp provide secondary target surfaces to identify adjuvant compounds that could be used in combination to more efficiently inhibit XRCC4/Ligase IV complex formation and DNA repair.
authors.affiliation: Chemical Biology Graduate Program, Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada, L8S 4M1; Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada, L8N 3Z5.
authors.forename: Meghan J
authors.initials: MJ
authors.lastname: McFadden
authors.forename: Wilson K Y
authors.initials: WK
authors.lastname: Lee
authors.forename: John D
authors.initials: JD
authors.lastname: Brennan
authors.forename: Murray S
authors.initials: MS
authors.lastname: Junop
mesh_terms: D001667:Binding, Competitive; D059766:DNA End-Joining Repair; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006801:Humans; D058990:Molecular Targeted Therapy; D009369:Neoplasms / Q000188:drug therapy; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D004268:DNA-Binding Proteins; D010446:Peptide Fragments; C101467:XRCC4 protein, human; D011088:DNA Ligases; D000072481:DNA Ligase ATP
keywords: NHEJ; inhibition; interaction surfaces; protein-protein interaction